DESCRIPTION: Virtually all eukaryotic organisms appropriately examined have been shown to possess the capacity for endogenous temporal control and organization known as a circadian rhythm. The cellular machinery responsible for generating this ability is collectively known as the biological clock. A healthy circadian clock underlies both physical and mental health. Because of the ubiquity of its influence on human mental and physiological process - from circadian changes in basic human physiology to the clear involvement of rhythms in work/rest cycles an sleep - understanding the clock is basic to prevention and treatment of many physical and mental illnesses. Our specific aims are designed to further our understanding of the means through which the clock regulates cat behavior in a tractable and well tuned model system and eventually in mammals. In previous work we show that the frequency gene encodes central components in the feedback loop comprising a circadian clock. We have determined that regulation of the expression of frq represents a major control point where external factors converge to affect the oscillator. In Specific Aim 1 we will study how transcriptional regulation of frq is effected. We will study proteins known to be required forfrq expression, appraise the importance of naturally occurring antisense frq transcripts, and execute a genetic screen to identify factors affecting frq expression. ->We have shown that the FRQ proteins are regulated both through synthesis and through modification, factor important in determining the kinetics of the clock cycle. In Specific Aim 2 we will study what regulates FRQ translation and how this responds to external factors including temperature. We will study FRQ phosphorylation, the relationship between phosphorylation and turnover, and will look for proteins with which FRQ interacts. We have established that the FRQ polypeptides are central components of the feedback loop comprising the clock. In Specific Aim 3 we will begin to analyze what FRQ regulates and both where and how it works. Our long term goals are to describe, in the language of genetics and biochemistry, the feedback cycle comprising the mechanism of the circadian clock, how this cycle is synchronized with the environment, and how tin information generated by the feedback cycle is used to regulate the behavior of cells. These projects a complementary and mutually enriching in that they rely on genetic and molecular techniques to dissect, and ultimately to understand. the organization of the cells as a function of time.